Optical system for a scanning fluorometer

ABSTRACT

A method and apparatus for determining the fluorescence, luminescence, or absorption of a sample is provided. The sample may either be contained within a cuvette or within one or more sample wells within a multi-assay plate. A combination of a broadband source, a monochromator, and a series of optical filters are used to tune the excitation wavelength to a predetermined value within a relatively wide wavelength band. A similar optical configuration is used to tune the detection wavelength. In one aspect, multiple optical fibers are coupled to the excitation source subassembly, thus allowing the system to be quickly converted from one optical configuration to another. For example, the source can be used to illuminate either the top or the bottom of a sample well within a multi-assay plate or to illuminate a single cuvette cell. Similarly, multiple optical fibers are coupled to the detector subassembly. In another aspect, the excitation light and the detected sample emissions pass to and from an optical head assembly via a pair of optical fibers. The optical head assembly is scanned across one axis of the sample multi-assay plate. The multi-assay plate is mounted to a carriage assembly that scans the plate along a second axis orthogonal to the first axis. In another aspect, an optical scanning head assembly is used that includes mirrored optics for coupling the excitation source to the sample and the emitted light to the detector.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of Provisional ApplicationSerial No. 60/096,999, filed Aug. 18, 1998 and a continuation-in-part ofU.S. patent application Ser. No. 09/274,753 filed Mar. 23, 1999.

This application is related to U.S. patent application Ser. Nos.09/274,792, 09/274,796, and 09/274,791.

FIELD OF THE INVENTION

The present invention relates generally to detection systems, and moreparticularly, to a method and apparatus for detecting fluorescence,luminescence, or absorption in a sample.

BACKGROUND OF THE INVENTION

In biology as well as other related scientific fields, samples areroutinely characterized by examining the properties of fluorescence,luminescence, and absorption. Typically in a fluorescence study,selected tissues, chromosomes, or other structures are treated with afluorescent probe or dye. The sample is then irradiated with light of awavelength that causes the fluorescent material to emit light at alonger wavelength, thus allowing the treated structures to be identifiedand to some extent quantified. The wavelength shift between the peakexcitation wavelength and the peak fluorescence wavelength is defined asthe Stokes shift and is the result of the energy losses in the dyemolecule.

In a luminescence study, the sample material in question is notirradiated in order to initiate light emission by the material. However,one or more reagents may have to be added to the material in order toinitiate the luminescence phenomena. An instrument designed to monitorluminescence must be capable of detecting minute light emissions,preferably at a predetermined wavelength, and distinguishing theseemissions from the background or ambient light.

In a typical light absorption study, a dye-containing sample isirradiated by a light source of a specific wavelength. The amount oflight transmitted through the sample is measured relative to the amountof light transmitted through a reference sample without dye. In order todetermine the concentration of dye in a sample, both the lightabsorption coefficient (at the wavelength used) and the path lengththrough the sample must be known. Other relative measurements may alsobe of interest, for example determining the wavelength dependence of theabsorption.

In general, an instrument designed to determine the fluorescence of asample requires at least one light source emitting at one or moreexcitation wavelengths and a detector for monitoring the fluorescenceemissions. This same instrument can often be used for both luminescenceand absorption measurements with only minor changes.

U.S. Pat. No. 4,626,684 discloses a fluorescence measurement system foruse with a multi-assay plate. The disclosed system uses concaveholographic gratings to control both the excitation and emissiondetection wavelengths. Optical fibers are used to couple the opticalscanning head to both the source and detector subassemblies. The pathsof both the excitation light and the fluorescent emissions areorthogonal to the surface of the material under study.

U.S. Pat. No. 4,501,970 discloses a fluorometer for use with multi-assayplates. The disclosed system directs the excitation beam of lightthrough the open top of the sample holding vessel and receives thefluorescent emission through this same opening. The system uses a seriesof mirrors and masks to decouple the excitation light from the emittedfluorescence, thereby reducing the noise signal level in the detectorand increasing the sensitivity of fluorescence detection.

From the foregoing, it is apparent that a high sensitivity, wavelengthscanning fluorometer is desired.

SUMMARY OF THE INVENTION

The present invention provides a method and apparatus for determiningthe fluorescence, luminescence, or light absorption of a sample. Thesample may either be contained within a cuvette or within one or moresample wells of a multi-assay plate. The system is designed toaccommodate a variety of different multi-assay plates in which the platedimensions as well as the number of sample wells varies.

In one aspect of the invention, an excitation means is provided foreither fluorescence or absorption measurements. The excitation meansincludes a broadband light source, a monochromator, and a series ofoptical filters. This combination of optical components allows theexcitation wavelength to be tuned to a predetermined value within arelatively wide wavelength band. Depending upon the dispersion of thecomponents, bandpass values of approximately 10 nanometers are commonlyachievable. A similar optical configuration is used to detect theemissions from the sample (i.e., fluorescence or luminescence) or theamount of light absorbed by the sample. The detection means includes aphotomultipler tube detector, a diffraction grating, and a series ofoptical filters.

In another aspect of the invention, multiple optical fibers are coupledto the excitation source, thus allowing the system to be quicklyconverted from one optical configuration to another. For example, thesource can be used to illuminate either the top or the bottom of asample well within a multi-assay plate or to illuminate a single cuvettecell. Similarly, multiple optical fibers are coupled to the detector.The multiple detector fibers allow the system to be easily convertedfrom detecting fluorescence or luminescence to detecting the amount ofexcitation light passing through the sample (i.e., for absorptionmeasurements). The multiple detection fibers also allow the opticalconfiguration to be converted to match the excitation configuration,e.g., cuvette cell versus multi-assay plate.

In another aspect of the invention, the excitation light and thedetected sample emissions pass to and from an optical head assembly viaa pair of optical fibers. The optical head assembly is coupled to a pairof guide rails and controlled by a step motor, thus allowing the headassembly to be driven along one axis of a multi-assay plate. Themulti-assay plate is mounted to a carriage assembly that is also coupledto a pair of guide rails and controlled by a step motor. The carriageassembly drives the multi-assay plate along a second axis orthogonal tothe first axis.

In another aspect of the invention, the system is designed toaccommodate a wide range of sample intensities automatically, such aswould be expected from a group of random samples within a multi-assayplate. In order to accommodate varying intensities, a photomultipliertube detector is used and the voltage is automatically varied in orderto change its gain. The automatic voltage adjustment is performed inthree steps, each providing a nominal dynamic range of three decades.Alternatively, the voltage adjustment can be performed in more thanthree steps employing finer gradations of dynamic range.

In another aspect of the invention for use with a multi-assay plateconfiguration, the system is designed to minimize the effects oftemperature drop from one sample to another that are due to evaporativecooling. Specifically, the plate holding carriage moves the multi-assayplate to a sample holding area between readings. Within the sampleholding area the multi-assay plate is confined by an upper or lidsurface that is close to the upper surface of the multi-assay plate. Thesides of the multi-assay plate may also be confined. When themulti-assay plate is within this area the relative humidity above theplate rises to more than 90 percent, thus reducing evaporative cooling.This aspect of the invention is preferably coupled to a temperatureregulation and air circulation system.

In another aspect of the invention, an optical scanning head assembly isused that includes mirrored optics for coupling an excitation source tothe sample and the emitted light to a detector. An ellipsoidal focussingmirror is used to magnify and focus the source light projected from anoptical fiber onto the sample. A portion of the source light isreflected by a beamsplitter onto a reference detector used to monitorthe output of the source. The light from the ellipsoidal mirror passesthrough an aperture in a second ellipsoidal mirror prior to impingingupon the sample. The light emitted by the sample within the sample well(e.g., fluorescence) is reflected by the second ellipsoidal mirror andimaged onto the entrance aperture of an optical fiber coupled to thedetector subassembly. The optical axes of both mirrors are slightlyoffset from the sample well normal. The offset minimizes the amount oflight reflected from the meniscus of the sample or the bottom surface ofthe sample well that enters the detection subassembly.

In another aspect of the invention, stray light reduction is optimizedin order to achieve improved sample emission detection uniformity,particularly emission detection uniformity for an array of fluorescencemeasurements of a specific sample well. In one approach, a wide-bandbandpass filter is used. In another approach, a dual excitationmonochrometer is used. In yet another approach, a dual emissiondetection monochrometer is used. In yet another approach, both a dualexcitation monochrometer and a dual emission detection monochrometer areused.

In another aspect of the invention, time tags are recorded for samplescontained within a multi-assay plate. The time tags can be used tomonitor compositional time dependent properties, for example thoseassociated with a kinetic reaction. The time tags can also be used toinsure that a comparison of individual samples within a multi-assayplate is accurate and is not biased by variations in the amount of timepassing between the steps of sample preparation and samplecharacterization. In one mode of time tagging, a time tag is recordedfor each critical preparation step and each critical characterizationstep for every sample of interest. In a second mode of time tagging,only a single time tag is recorded for the entire multi-assay plate foreach critical step of either preparation or characterization. In thismode, however, every sample of the multi-assay plate is sequentiallyprepared or characterized with a set interval passing between thepreparation or characterization of successive samples.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates the detection system of the presentinvention;

FIG. 2 is an illustration of the outer casing of one embodiment of theinvention;

FIG. 3 is an illustration of the combined optical subassemblies;

FIG. 4 is a perspective view of an excitation filter wheel;

FIG. 5 is a perspective view of an excitation filter wheel assembly;

FIG. 6 is an exploded view of a shutter assembly;

FIG. 7 is an illustration of the combined shutter plates utilized in theshutter assembly shown in FIG. 6;

FIG. 8 is an illustration of a PMT housing and slit;

FIG. 9 is a perspective view of an emission filter wheel assembly;

FIG. 10 is an illustration of an emission filter wheel, includingfilters;

FIG. 11 is an illustration of a multi-assay plate carriage assemblyaccording to the invention;

FIG. 12 is an illustration of a scanning optical stage assemblyaccording to the invention;

FIG. 13 is an illustration of the combined carriage and optical stageassemblies;

FIG. 14 is an illustration of a portion of the temperature controlsystem used with the present invention;

FIG. 15 is an illustration of the underside of the base assembly of thepreferred embodiment of the invention;

FIG. 16 is a block diagram of the detection scheme in the preferredembodiment of the invention;

FIG. 17 is a block diagram outlining the wavelength optimizationprocedure;

FIG. 18 illustrates the algorithm used when a plate is read using theautomatic mode of the invention;

FIG. 19 illustrates an alternative approach to the technique shown inFIG. 18;

FIG. 20 illustrates a variation of the method illustrated in FIG. 18;

FIG. 21 illustrates another approach that may be utilized by the presentinvention;

FIG. 22 illustrates a slight variation of the method shown in FIG. 21;

FIG. 23 illustrates a spectrum mode of analysis for use with theinvention;

FIG. 24 schematically illustrates the well optics;

FIG. 25 is an exploded view of an optical scanning head according to thepreferred embodiment of the invention;

FIG. 26 is a perspective upper view of the optical scanning head shownin FIG. 25;

FIG. 27 is a perspective lower view of the optical scanning head shownin FIGS. 25 and 26;

FIG. 28 is a detailed view of the apertured detection mirror used in thepreferred embodiment of the optical scanning head;

FIG. 29 is an illustration of an alternative optical configuration foruse with a sample well;

FIG. 30 is an illustration of an alternative optical configuration foruse with a cuvette cell;

FIG. 31 illustrates the relationship between the position of theexcitation light in the sample well with the amount of light reflectedinto the detector fiber;

FIG. 32 is an illustration of the artifacts that can be generated duringwell-scanning;

FIG. 33 is an illustration of the artifacts that can be generated duringwell-scanning after an additional excitation light filter is insertedbetween the light source and the sample wells 125;

FIG. 34 schematically illustrates an alternate embodiment of a scanningfluorometer according to the present invention;

FIG. 35 schematically illustrates yet another alternate embodiment of ascanning fluorometer according to the present invention;

FIG. 36 is a schematic illustration of the principal components of atime tagging system according to at least one embodiment of theinvention;

FIG. 37 illustrates the methodology associated with the embodiment shownin FIG. 32; and

FIG. 38 illustrates the methodology associated with an alternative timetagging embodiment.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

System Overview

FIG. 1 schematically illustrates the principal components of at leastone embodiment of a scanning fluorometer system 100 according to thepresent invention. Preferably system 100 is constructed utilizingsubassembly modules. This module approach offers several benefits.First, it allows a non-functioning subassembly to be easily removed andreplaced with a functioning subassembly, thereby minimizing the amountof time that the system is inoperable. Second, modules can be replacedor augmented as either the user's requirements change, or as improvedsubassemblies become available, thus providing for system growth. Third,this approach allows for increased on-site calibration and/ormaintenance.

A light source subassembly 101 within system 100 generates illuminationof a predetermined wavelength. Preferably the source for subassembly 101is a broadband source, such as a xenon flash lamp 103. The light fromlamp 103 may pass through one or more apertures 105 in order tocondition the light before passing through an optical filter 107 mountedin an opening of a filter wheel 108. The wavelength of light emitted bysource subassembly 101 is determined by a combination of filter 107, amovable grating 109, and apertures formed by the input apertures ofoptical fibers 119.

The light from source subassembly 101 is used to either illuminate awell 125 of a multi-assay plate 111 contained within a multi-assay platechamber subassembly 113 or a cuvette 115 within a cuvette chambersubassembly 117. Multi-assay plate 111 is retained by a holding fixture.The light from source subassembly 101 is transmitted to multi-assayplate chamber subassembly 113 or cuvette chamber subassembly 117 via aselected fiber of optical fibers 119. Furthermore, the source light canbe transmitted either through the open top portions 124 of wells 125 orthrough transparent closed bottom portions 126 of wells 125, theselection of which is determined by the particular optical fiber 119selected to couple source subassembly 101 to multi-assay plate 111. Anoptical shutter 121 within source subassembly 101 establishes which offibers 119 receives light from source 103. One or more focussing mirrors123 focus the light passing through fibers 119 into the chamber ofinterest, i.e., multi-assay plate well 125 or cuvette 115.

The light, either from cuvette 115, top portion 124 of well 125, orbottom portion 126 of well 125, is collected with optics 127. Thecollected light can be either light emitted as fluorescence orluminescence, or transmitted light used for an absorption measurement.The collected light passes through a selected optical fiber of fibers129 to a detection subassembly 131. When transmitted light is used foran absorption measurement in wells 125, the preferred configuration isto pass the light first through top portion 124 of wells 125, thenthrough the sample materials contained within wells 125, and finallythrough the bottom portion 126 of wells 125. During absorptionmeasurements, the transmitted light is collected by optics 127positioned under multi-assay plate 111. The collected light is thenfocused onto a selected fiber 129 for transmission to detector 135 indetector subassembly 131. In a first alternative configuration used forabsorption measurements, as in the above configuration the light enterswell 125 through top portion 124. After the light passes through thesample materials within well 125, however, it is reflected back by amirror underneath of the sample well (not shown) and collected by opticspositioned above the well (not shown). In a second alternativeconfiguration a detector, preferably a photodiode, is located directlyunder the well (not shown) and collects the light transmitted throughwell 125 and the sample materials contained therein. In a thirdalternative configuration (not shown) the light enters well 125 throughbottom portion 126, passes through the sample materials, passes throughtop portion 124, and is then collected and focussed onto a detector. Inthis configuration the detector may either be mounted remotely or bemounted in close proximity to top portion 124.

A shutter 133 determines which fiber 129 is monitored by subassembly131. The light from a selected fiber 129 is focussed onto a detector 135by a movable, focussing grating 137. Preferably detector 135 is aphotomultiplier tube (i.e., PMT). The light may pass through one or moreapertures 141 to reduce stray light before impinging on detector 135.The combination of grating 137, aperture 141, and a filter 139 mountedin an opening of a filter wheel 140 determines the wavelength of lightdetected by detector 135.

Grating 109 allows the excitation wavelength to be continuously variedover a relatively wide wavelength band. Similarly, grating 137 allowsthe detection wavelength to be continuously varied over a wide range ofwavelengths. In the preferred embodiment of the invention, gratings 109and 137 each have a focal length of approximately 100 millimeters, thusallowing excitation subassembly 101 and detection subassembly 131 to berelatively compact. As the gratings are preferably holographic gratingswith 1200 grooves per millimeter, the dispersion of the gratings withthis focal length provides a nominal 10 nanometer bandpass. In apreferred embodiment of the invention, the blaze angle of the gratingsis 500 nanometers. However, the gratings may be blazed at differentangles, thus further enhancing the decoupling of the excitation andfluorescence wavelengths. Preferably the arc of lamp source 103 isfocused onto the entrance aperture of fiber 119.

FIG. 2 illustrates the outer casing of one embodiment of the invention.In this embodiment a multi-assay plate 111 that is ready for testing isplaced within reading chamber 202 of instrument 203 via a housing door205. In at least one embodiment of the invention, the instrument canalso be used to test a cuvette, preferably by inserting the cuvette intoa cuvette port 209. A control panel 211 provides a user interface,allowing the user to initiate testing as well as set various testingprotocols. Preferably control panel 211 also includes a simple readoutsystem such as a LCD readout, thus providing the user with positiveindications of selections as well as status.

Instrument 203 is preferably coupled to a data processing system 213 viaa cable 215. Data processing system 213 is used to manipulate the data,store the data, and present the data to the user via either a monitor217 or a printer 219. Depending upon the system configuration,processing system 213 can also be used to control the test itself (i.e.,test initiation, test protocol settings, etc.). In the preferredembodiment, an internal processor controls at least the basic testparameters by controlling movable gratings 109 and 137, filter wheels108 and 140, shutters 121 and 133, and the relative movement ofmulti-assay plate 111 to excitation optics 123 and detection optics 127.

FIG. 3 is an illustration of the optical subassemblies of the preferredembodiment of the invention. In order to decrease the overall size ofsystem 100, in this embodiment of the invention both source subassembly101 and detection subassembly 131 are contained on a single opticalbench 301.

Source 103 is mounted within a bracket 303 and coupled to a source highvoltage power supply 305. Preferably source 103 is a xenon flash lampdue to its relatively wide emittance wavelength band, ranging from theultraviolet to the infrared. In alternative embodiments, source 103 canbe a mercury arc lamp, a laser, an incandescent lamp (e.g., a tungstenlamp), or other source. The light from source 103 passes through afilter wheel 108 containing a plurality of optical filters 107. Filters107 can be bandpass filters (i.e., pass a band of wavelengths), cutofffilters (i.e., only pass wavelengths above or below a predeterminedwavelength), or any other type of optical filter that can be used tocontrol the wavelength of light passing through the filter and impingingon optical grating 109. The position of filter wheel 108, and thereforethe selected filter in the excitation beam path, is controlled by motor309. A position sensor 311 (e.g., an optical switch) or other means isused to determine the position of wheel 108, and thus the filter 107within the beam path. Preferably motor 309 and position sensor 311 arecoupled to a controller internal to the instrument.

The light passing through the selected filter 107 in filter wheel 108 isreflected off of grating 109. Grating 109 is coupled via a shaft 313 toa fine control motor, such as a stepper motor, that is used to set theangle of grating 109 with respect to the incident light beam. Grating109 focuses the light onto a shutter assembly 315. Covering excitationsource assembly 101 is a cover 317, thus helping to minimize sourcelight inadvertently entering the system.

FIG. 4 is a perspective view of filter wheel 108 and filters 107 andFIG. 5 is a perspective view of the filter wheel assembly includingwheel 108, motor 309, and position sensor 311. In an embodiment of theinvention, filter wheel 108 contains 5 long pass filters covering thewavelength bands shown in Table 1 below. Excitation filters 107 serve adual purpose. First, the filters are used to block the second order,thereby reducing stray light. Second, a higher number of filters areused than is common so that the cutoff may be placed as close aspossible to the selected wavelength, thus blocking higher orders andhelping to reduce stray UV light from exciting background fluorescencein the sample. Preferably the cutoff wavelength (i.e., the wavelength of50% nominal transmission of the long pass filters) is approximately 10nanometers below the selected wavelength. Light wavelengths less thanthe cutoff wavelength are substantially blocked while longer wavelengthsare substantially transmitted to excitation grating 109.

TABLE 1 Endpoint and Kinetic Cutoff Selection Modes Filter No. λ (nm)Excitation λ (nm) 1 No Filter 200-330 2 320 330-410 3 395 410-510 4 495510-610 5 590 610-710 6 695 710-800

An exploded view of shutter assembly 315 is shown in FIG. 6. Shutterassembly 315 is used to control the coupling of the light beam focussedby grating 109 onto one or more optical fibers. In the preferredembodiment, a single fiber is mounted within a fiber mounting opening601 in a bracket 603.

In an alternate embodiment, multiple fibers are mounted within bracket603, shutter assembly 315 controlling which, if any, of the fibersreceive light from grating 109. In the alternate embodiment, shutterassembly 315 includes an inner shutter plate 605 and an outer shutterplate 607. Shutter plates 605 and 607 are coupled via a bushing 609 to amounting hub bracket 611. As illustrated, shutter plates 605 and 607 areplaced into the desired location and locked onto hub bracket 611 via aset screw 613. Alternatively, hub bracket 611 may be replaced with amotor, the motor allowing the position of the shutter plates to beremotely controlled.

FIG. 7 is an illustration of combined shutter plates 605 and 607 showingthe alignment of the apertures of the two plates. As shown, the distance701 between each set of aligned apertures and the center of rotation 703varies. Therefore as the combined shutter plates are rotated along axis703, the position of the light beam passing through the shutter assemblychanges. Given the shutter assembly illustrated in FIGS. 6 and 7, threefibers could be co-located within bracket 603, combined shutter plates605 and 607 determining which of the fibers receive light from theexcitation source. As illustrated, shutter plates 605 and 607 have threeapertures thereby limiting the total number of fibers that can becoupled to the excitation source through this assembly to three.However, the number of apertures and thus the number of fibers areprimarily limited by the size of the light beam focussed by grating 109onto the shutter assembly. Furthermore, by varying the aperture sizes ofshutter plates 605 and 607, the size of the passed beam can also becontrolled.

The light from multi-assay plate chamber subassembly 113 or cuvettechamber subassembly 117 enters detection subassembly 131 via a shutterassembly 319. The design of shutter assembly 319 is substantially thesame as shutter assembly 315 and includes a pair of shutter plates 321,a bushing 323, and a hub bracket 325. Although in the illustratedembodiment there is only a single fiber opening 327, bracket 325 couldinclude multiple fiber openings thereby allowing shutter plates 321 tocontrol which fiber passes light through to the detector. Additionallyand as described above, shutter plates 321 can also be used to controlthe size of the beam simply by varying the size of the apertures.

Light passing through shutter plates 321 is focussed by grating 137 ontodetector 135. Grating 137 is coupled to a high accuracy motor via acoupler 329. The combination of rotatable grating 137 and a filterassembly 331 determines the detected light wavelength. Due to thelocation of filter assembly 331 between grating 137 and detector 135,native fluorescence in filter 139 is substantially eliminated. Theentire detection subassembly is enclosed with a cover 333 that isattached to optical bench plate 301. A gasket 335 insures a light sealbetween cover 333 and plate 301. Similarly gaskets 337 insure a lightseal between cover 333 and optical fiber 129 coupled to shutter assembly319 via opening 327. Cover 333 as well as the various gaskets (e.g., 335and 337) substantially reduce the amount of stray light enteringdetector 135. As illustrated in FIG. 8, attached to the cover ofdetector 135 is slit aperture 141 that also enhances the rejection ofstray light in the present invention.

Filter assembly 331 is shown in greater detail in FIGS. 9 and 10. Asshown in FIG. 9, emission filter assembly 331 includes a bracket 901, afilter wheel 140, a stepper motor 905, and a filter wheel positionsensor 907 such as an optical sensor. Motor 905 is used in conjunctionwith position sensor 907 and either an internal or external processor toplace the appropriate filter 139 in the optical beam path. Asillustrated in FIG. 10, filter wheel 140 includes 15 filters 139 as wellas an open filter space 1001. In different embodiments filter wheel 140may include fewer or greater numbers of filters. Filters 139 can bebandpass filters, cutoff filters, or any other type of optical filterthat can be used to control the wavelength of light impinging ondetector 135. In the preferred embodiment of the invention filters 139are long pass cutoff filters.

As previously noted, the present invention can be used to measurefluorescence, luminescence, or absorption. In the fluorescence mode, theprincipal limiting factor in achieving high sensitivity is thebackground. A major contributor to the background is stray light fromthe excitation source. A portion of this stray light is due to the closeproximity of the excitation and fluorescence emission wavelengths andthe difficulty associated with preventing the excitation sourcewavelengths from passing through the optical assembly and into thedetector. Another portion of the stray light is due to light thatfollows a non-direct, non-intentional path into the detector, e.g.,reflections off of mounting brackets. The present invention limits thelatter type of stray light by enclosing both the excitation anddetection subassemblies 101 and 131, respectively, and using opticalfibers to couple the various optical assemblies.

In a typical fluorometer, the band of wavelengths used to excite thefluorescent material is separated from the band of wavelengths passed tothe detector by using monochrometers having prisms, or gratings, forwavelength dispersion and an exit slit to select the wavelength ofinterest or by using optical filters. Unfortunately neither a gratingnor an optical filter is capable of absolutely eliminating the passageof undesired wavelengths. In addition, since the intensity of theexcitation source is typically at least six orders of magnitude greaterthan the intensity of the fluorescent emissions, neither gratings noroptical filters alone can provide the desired sensitivity since somesmall fraction of in-band source light still reaches the detector. Thepresent invention uses optical filters 107 and 139 in conjunction withboth an excitation and an emission monochrometer having, respectively,gratings 109 and 137. The combination of filters and gratings furtherdecreases the amount of source light outside of the excitationwavelength band, and thus within the emission wavelength band, reachingthe detector. As a result, the present invention is capable of extremelyhigh detection sensitivities.

In order to achieve high sensitivity the present invention also utilizesboth apertures and absorbing beam blockers. For example, shutters 321substantially limit the light reaching grating 137 to light exiting aselected one of optical fibers 129. In addition, slit 141 limits theviewing angle of detector 135 to grating 137. Similarly, apertures andbeam blockers are used in excitation subassembly 101 to limit the lightilluminating grating 109 as well as to limit the viewing angle of theexit aperture (formed by each entrance slit of optical fibers 119).

Although the combination of optical filters and gratings is capable ofachieving improved sensitivity through the reduction in stray light,this combination may also reduce the level of light in the desiredwavelength band to an unacceptable level. In order to avoid thisproblem, the invention preferably uses at least 15 different longwavelength pass emission filters 139. By using a large number offilters, the difference in cutoff wavelength for adjacent filters issmall in comparison to the typical minimum Stokes shift. For example,assuming a desired wavelength band of approximately 400 to 700nanometers, 16 filters with a nominal 20 nanometer spacing in cutoffwavelength can be used.

Due to the combination of optical filters and gratings in the presentinvention, the system is preferably capable of operating in severaldifferent modes, thus insuring that the best performance for aparticular sample is achieved. Specifically, the user is able to set thesystem to operate either in a manual mode or in an automated mode. Inthe latter mode the user can select the system to provide eitherexcitation priority or emission priority.

Preferably automated emission priority mode is the default settingexcept when the user is operating in the emission scan mode. In theautomated emission priority mode the system compares the selectedemission wavelength of emission grating 137 to the 50 percenttransmission point for each of the emission filters 139. The system thenselects a long pass filter offering a cutoff wavelength (e.g., 50percent maximal transmission wavelength) as shown in Table 2 below.Table 2 consists of all of the possible emission wavelengths selectableby emission grating 137 and the corresponding filters (as indicated bytheir cutoff wavelengths) which are selected automatically. For example,if a user selects an emission wavelength of 500 nanometers, the systemwould review the look-up table which indicates that the cutoffwavelength for the selected filter is 495 nanometers. At emissionwavelengths below 415 nanometers, no emission filter is selected.

TABLE 2 Automatic Cutoff Endpoint and Kinetic Selection Modes Filter No.λ (nm) Emission λ (nm) 1 None <415 2 420 415-434 3 435 435-454 4 455455-474 5 475 475-494 6 495 495-514 7 515 515-529 8 530 530-549 9 550550-569 10  570 570-589 11  590 590-609 12  610 610-629 13  630 630-66414  665 665-694 15  695 695-900

As noted above, in the preferred embodiment of the invention theemission priority mode is the default as it provides the bestperformance for most assays. The exception to this default is inemission scanning. In emission scanning mode no emission filters 139 areautomatically utilized because this would mean filter changes duringscanning, resulting in unwanted discontinuities. Cutoff filters (asingle choice per emission scan) may be added manually for optimizationpurposes. In excitation scanning mode, emission filters 139 areautomatically selected according to Table 2.

In the alternative excitation priority mode, the system compares theselected excitation wavelength to the cutoff wavelength for each of theemission filters 139. The system then selects the filter offering acutoff wavelength that is both nearest to and greater than (inwavelength) the selected excitation wavelength. For example, if a userselects an excition wavelength of 400 nanometers, and assuming thefilter preformance disclosed in the previous example, the system wouldselect the 420 nanometer cutoff filter. This filter has a 50 percentmaximal transmission wavelength at 420 nanometer and greatertransmission at longer wavelengths.

Scanning Assembly

FIGS. 11-15 illustrate various aspects of the sample scanning assemblyof the invention. Although in at least one embodiment of the inventionthe system is used to read a cuvette using cuvette port 209, the primaryapplication for this invention is reading multi-assay plates.Furthermore the preferred embodiment of the invention is designed to beadaptable to multi-assay plates of varying configurations (i.e., varyingquantities and sizes of sample wells, various plate sizes, etc.).

In FIG. 11, a sample plate holding fixture 1101 is designed toaccommodate multi-assay plates of standard dimensions (e.g., 86 by 129millimeters). In order to accommodate other sized plates, an adaptorplate (not shown) is mounted within fixture 1101, the non-standard platefitting within the adaptor plate. Fixture 1101 supports the multi-assayplate or adaptor plate along the edges using a support frame 1103. Thusan area 1105 immediately under the sample wells of the multi-assay plateremains open, allowing a variety of sample measurements to be made thatrequire access to both the upper and lower surfaces of the sample wells.Holding fixture 1101 slides along a pair of railings 1107 that aremounted to a base assembly 1109. A drive motor 1111 moves fixture 1101and thus the multi-assay plate along a first axis parallel to railings1107 by using a belt and pulley system 1113.

FIG. 12 is an illustration of the optical scanning assembly 1200.Scanning assembly 1200 allows an optical head 1201 to be scanned along asecond axis perpendicular to railings 1107. Thus scanning assembly 1200,used in conjunction with scanning fixture 1101, allows an optical head1201 to be scanned in two dimensions, thereby providing a means ofanalyzing each well of a two-dimensional array of wells within amulti-assay plate.

In the preferred embodiment of the invention, optical head 1201 includesthe optics required to illuminate the sample as well as the opticsnecessary to gather the emitted light. Optical fibers 119 and 129,although not shown in this illustration, are coupled to optical head1201 via a strain relief bracket 1203. Optical head 1201 slides along apair of railings 1205 that are mounted to a bottom assembly plate 1207via a pair of brackets 1209. A drive motor 1211 and a belt and pulleysystem 1213 move optical head 1201 along a second axis orthogonal to thefirst axis. FIG. 13 is an illustration of optical scanning assembly 1200mounted to base assembly 1109. Underlying multi-assay plate holdingfixture 1101 is also shown in this figure.

In the preferred embodiment of the invention, scanning motors 1111 and1211 are both under the control of an internal processor. Typicallyprior to use, the user inputs the sample plate configuration (e.g., howmany wells, plate type, well size, etc.). The user then programs theinternal processor to scan the designated sample plate utilizing one ofa variety of scan modes. For example, an on-the-fly scanning mode can beused in order to minimize the amount of time it takes to read a sampleplate by eliminating the acceleration and deceleration times. In thismode fixture 1101 and optical scanning head 1201 are scanned in acontinuous fashion, for example utilizing a zig-zag pattern. Source 103is flashed as optical head assembly 1201 passes over each well, thusallowing a single measurement to be made for each well. Alternatively,processor 1101 can be programmed to place optical head 1201 over eachwell for a predetermined period of time, allowing a predetermined numberof sample readings (initiated by flashes emitted by source 103) to bemade for each well.

Many fluorescence, luminescence, and absorption measurements areextremely sensitive to outside environmental factors such astemperature. This effect can become an even greater problem as thenumber of sample wells per multi-assay plate increase, leading tovariations across the plate.

One approach to overcoming the environmental problem is to simplycontrol the temperature of the reading chamber. This approach, however,may do little to minimize the effects of temperature drop caused byevaporative cooling. A second approach is to combine temperature controlwith the use of a multi-assay plate cover. Although the cover minimizesevaporative cooling and allows for temperature control, it alsotypically leads to a degradation in instrument sensitivity due to theeffects of the lid on the optical system (i.e., increased stray lightdue to cover scatter, absorption by the lid, etc.).

The present invention overcomes these problems through the use of avirtual lid in combination with a temperature control system. In thepreferred embodiment, carriage 1101 moves multi-assay plate 111 to anarea 1115 between system readings. A lid 1401 is directly above thisarea. Additional members, for example made of foam, can also be used tofurther enclose the multi-assay plate when it is in area 1115. Thebottom plate 1207 of optical head 1201 rests on the top side of lid 1401(i.e., opposite to reading chamber 202). An opening 1208 exists in bothbottom plate 1207 and lid 1401, thus allowing excitation and emissionlight to pass from the optical head to the samples contained in themulti-assay plates within chamber 202. Preferably the dimensions ofopening 1208 are about 1.2 millimeters by 104 millimeters. When carriage1101 moves multi-assay plate 111 below lid 1401, the lid surface isapproximately 10 millimeters above the surface of the multi-assay plateand the sides of the multi-assay plate are tightly confined. As such,once the multi-assay plate is moved into resting position 1115 and theaccess door 1116 has been closed, the humidity above the plate rises tomore than 90 percent, thus reducing evaporative cooling. This systemreduces the variations from sample well to sample well within amulti-assay plate to preferably less than ±0.20° C., and generally toless than ±0.50° C.

In the illustrated embodiment, variations in multi-assay plate size areaccommodated by using various adaptor plates. The adaptor plates notonly insure that the multi-assay plate fits support frame 1103, they canalso be used to insure that the top of the multi-assay plate issufficiently close to the surface of lid 1401 to minimize temperaturevariations between the wells. In an alternate embodiment, the relativedistance between lid 1401 and the top of a multi-assay plate in carriageassembly 1101 can be optimized by adjusting either the vertical positionof the lid or the carriage assembly carrying multi-assay plate 111. Inthis embodiment either the lid or the carriage assembly is coupled to amotor, the motor under the control of the internal processor. Preferablya sensor (e.g., optical sensor, mechanical position sensor, etc.) isused in conjunction with this motor and processor 1101 to control theseparation between the multi-assay plate and lid 1401. Alternatively,the user can input the type of multi-assay plate in use and the internalprocessor can use a look-up table to determine the amount of adjustmentnecessary for the type of multi-assay plate in use.

In order to control the temperature of area 1115 as well as the rest ofreading chamber 202, one or more heaters 1403 are attached to variousportions of the reading chamber. Preferably heaters 1403 are attached tolid 1401 as shown. One or more temperature monitors (e.g., thermistors)1405 are used to monitor the temperature of the reading chamber. Anouter cover 1407 is coupled to lid 1401 to facilitate temperaturecontrol within this area. Other covers such as an internal cover 1409and an outer cover 1411 enclose the remaining upper portion of thereading chamber, thus further aiding in controlling the temperature ofthe system.

FIG. 15 is an exploded view of the underside of base assembly 1109.Attached to base assembly 1109 is an air circulation enclosure 1501. Afan 1503 forces air through enclosure 1501. The air passes throughperforations 1117 within the raised portions 1118 of base structure 1109as shown in FIG. 11. The air circulation system, including perforations1117, insure temperature uniformity throughout the reading chamberwithout causing undue air movement above the multi-assay plate.

Detection System

The present invention can be used in a variety of different modes,including end-point, kinetic, spectrum, and excitation or emissionwavelength scanning. Furthermore, the system can be used with eithermulti-assay plates or single cuvettes. One of the many advantages of thepresent invention is the ability to scan either or both the excitationwavelength and the detected wavelength using gratings 109 and 137 aswell as filters 107 and 139. This capability allows a user to determineaccurately not only the intensity of a detected emission, but also itssource (e.g., expected fluorophore, spectral convolution due to multiplefluorophores, background, etc.). Furthermore, the tunability of theexcitation and emission wavelengths allow both the excitation wavelengthand the detected emission wavelengths to be optimized while minimizingbackground detection, thereby achieving optimal instrument performance.

The principal difficulty in measuring fluorescence in a multi-sampleformat such as a multi-assay plate is the range of sample intensitiestypically encountered, often covering six orders of magnitude. Inaddition, if the detector is not optimized for each reading, it mayeither be saturated or operating below its peak sensitivity. Lastly, thefluorescence of each sample of the entire multi-assay plate must bequickly determined or the instrument is of limited use.

The present invention solves the above problems by using aphotomultiplier tube (i.e., PMT) as detector 135 and, in at least oneembodiment, automatically varying the voltage of the PMT detector inorder to change its gain. Preferably the automatic voltage adjustment isperformed in three steps, each providing a nominal dynamic range ofthree decades. Each time the voltage is changed there is a delay,typically between 0.1 and 10 milliseconds, to provide sufficient timefor detector stabilization. If the samples loaded into the multi-assayplate are highly random and exhibit large variations in molarconcentration, the system will have to undergo many voltage changes thusincreasing the plate reading time. This effect is most severe when theinstrument is operating in the on-the-fly scanning mode in which eachsample well is irradiated with a single flash of source light, typicallyon the order of a 3 microsecond flash. In this mode, depending upon thesystem configuration, if a voltage change is necessary either theindividual well or the entire column must be re-read.

In the preferred embodiment of the invention the user is able to selecteither an automatic mode of operation or a manual mode of operation. Inthe manual modes of operation the PMT detector voltage is set manually,thus avoiding the time delays that can be encountered when the systemautomatically controls the detector voltage. In one manual mode, theuser sets the detector voltage low enough to avoid detector saturationduring any reading. Unfortunately this voltage setting may sacrificedetector sensitivity. In another manual mode, the user sets the detectorvoltage high enough to maximize sensitivity. In this case readings fromsample wells containing high concentrations of analyte will be saturatedand therefore invalid. Therefore the capability of selecting from thedifferent modes of operation allows the user to select the highestpriority characteristic; wide dynamic range, minimal measurement time,or saturation avoidance. The various modes of operation are described infurther detail in a later section of the specification.

FIG. 16 is a block diagram of the preferred embodiment of the detectioncircuit. In this system an internal processor 1601, preferably internalto instrument 100, controls the various aspects of the system as well ascalculating the fluorescence for each sample. It is understood thatprocessor 1601 may, in fact, utilize several processors to perform thevarious functions of the invention. Processor 1601 controls the gain ofPMT detector 135 by controlling the output of the PMT high voltage powersupply 1603 via a digital to analog converter 1605. In the preferredembodiment of the invention, processor 1601 receives information fromsensor 311 and controls filter wheel 108 via motor 309 and similarlyreceives input from sensor 807 and controls filter wheel 140 via motor905. In addition, processor 1601 sends signals to the motors controllingthe rotation of gratings 109 and 137 in order to control the wavelengthof the excitation and emission monochromators. If shutter slits 605/607and 321 are coupled to stepper motors, these motors can also becontrolled by processor 1601. Preferably processor 1601 also controlsthe motion of the sample plate scanning motors.

Processor 1601 controls the output of source 103 by controlling when thesource is triggered. As illustrated in the FIG. 16 block diagram, theoutput of source 103 irradiates a sample 1607, the emitted fluorescencefrom sample 1607 being detected by detector 135. The current signalgenerated by PMT detector 135 is integrated by an op-amp/integratingcapacitor 1609. The charge level (i.e., voltage) is measured before andafter each flash of source 103 and the difference (i.e., PMT signal) iscalculated. The output signal from op-amp/integrating capacitor 1609passes to multiple gain amplifiers 1615-1618. Although the preferredgain amplifier only includes four gain amplifiers, it is understood thatboth fewer and greater numbers of gain amplifiers can be used in thepresent invention. A multiplexer 1611 selects one output to be sent toan analog to digital converter 1613, thereby providing four levels ofconversion gain. This same conversion is also performed for adark-current correction, thus allowing the integrated value of the PMTdark current to be subtracted from the readings without the occurrenceof a flash.

Processor 1601 adjusts PMT power supply 1603 by first measuring a lowreference that is contained within a sample well 1121 of carriage 1101as shown in FIG. 11. Preferably the low reference is a piece ofpolystyrene. This measurement is used to adjust power supply 1603 sothat highest gain ADC 1618 (i.e., 64×) is near a maximum value. Twoadditional lower PMT voltage levels are then selected so that the gaindynamic range provided by the multiple ADC conversions 1615-1618 overlapwhen the lower voltage levels are selected. In this manner when areading at ADC gain 1615 (i.e., 1×) approaches a maximum count, thereading at the high ADC gain level 1618 (i.e., 64×), decremented to thenext voltage level, results in a reading slightly lower than the maximumcount.

Each sample well of multi-assay plate 111 can be irradiated severaltimes, the results of separate readings (i.e., each “reading” comprisesirradiation and signal measurement steps) being averaged to provide anaverage sample well reading. Due to the PMT dark current characteristic,a targeted precision level can be achieved by averaging fewer readingsat higher signal levels than would be required at lower signal levels.Therefore samples with different sample intensities may be irradiated adifferent number of times where the number of readings is inverselyproportional to the square root of the sample intensity.

When used as a fluorometer for end-point or kinetic measurements, thepreferred embodiment of the invention first optimizes the selectedexcitation and emission wavelengths by using the procedure illustratedin FIG. 17. The first step is to select an emission wavelength betweenabout 20 and 50 nanometers, and preferably about 35 nanometers, higherin wavelength than the expected emission peak (step 1701). Theexcitation wavelength is then scanned up to within approximately 20nanometers of the fixed emission wavelength (step 1703). The observedpeak excitation is determined from this scan (step 1705) and theexcitation wavelength is fixed at this wavelength in at least oneembodiment of the invention (step 1707). Alternatively, the excitationwavelength may be fixed at the leading edge of this wavelength, atapproximately the 90 percent maximum. The latter approach is optimalwhen the difference between the excitation and emission peaks is small,i.e., less than 50 nanometers. The emission wavelength is then scanned(step 1709) and the observed peak emission wavelength is determined(step 1710). Next, the emission filter is selected (step 1711). Althougha default filter can be selected as previously described, in thepreferred embodiment the emission filter is selected by trial and error,thus requiring the repetition of steps 1709-1711 as shown. In thepreferred approach, step 1711 (i.e., selection of the cutoff emissionfilter) is achieved through comparison of the spectrum obtained from twosamples representing “signal” (i.e., signal due to a high concentrationfluorophore) and “background” (i.e., signal from a sample with nofluorophore) and selection is based on the optimization of thesignal-to-background ratio. Still another alternative is to use thedefault filter selected as previously described.

In the preferred embodiment of the invention, step 1711 is performedusing filter wheel 140. Filter wheel 140 includes the available emissioncutoff filters 139. Optimally the cutoff value (i.e., the wavelengthgiving 50 percent maximal light transmission) should be near the maximalemission wavelength, preferably between the excitation wavelength andthe maximal emission wavelength, but at least 35 nanometers greater thanthe excitation wavelength. In the preferred embodiment, the cutofffilter values are 420, 435, 455, 475, 495, 515, 530, 550, 570, 590, 610,630, 665, and 695 nanometers. Preferably there is one filter position onboth filter wheels 140 and 108 that is left open, i.e., with no filterin place, thus providing unlimited light transmission.

After the emission filter is fixed (step 1712), the emission wavelengthis fixed at a wavelength approximately 10 nanometers greater than theemission filter cutoff (step 1713). Assuming multiple samples containedin the sample wells of a multi-assay plate, each sample well is thenread using the excitation and emission wavelengths selected during theoptimization process (step 1715).

In a typical fluorometer, the detected fluorescence is not simply afunction of the quantity of the fluorescing material. Rather, thedetected fluorescence is also affected by detector sensitivity, detectordrift, source intensity (unless the source is referenced), volume of thesample that is excited (i.e., beam size and path length), andmonochromator and/or filter efficiency. In order to compensate for thesefactors, the present invention uses a pair of built-in fluorescencereferences referred to herein as a high reference and a low reference.The high and low references are 1123 and 1121, respectively, included ina portion of carriage 1101 as shown in FIG. 11. The low reference is aclean piece of polystyrene.

In the present invention, fluorescence is calculated in terms ofRelative Fluorescence Units (RFU). A RFU is defined as:${RFU} \equiv \frac{\left( {{PMT}\quad {{cal}.\quad {coeff}.}} \right)\quad \left( {{PMT}\quad {signal}} \right)\quad \left( {{Ref}.\quad {sens}.\quad {coeff}.} \right)}{\left( {{Filter}\quad {trans}} \right)\quad \left( {{Ref}.\quad {signal}} \right)\quad \left( ^{{PMT}\quad {voltage}} \right)\quad \left( {{PMT}\quad {{sens}.\quad {coeff}.}} \right)}$

where:

PMT cal. coeff.—The PMT calibration coefficient is determined bymeasuring the high reference at specific excitation and emissionwavelengths and at constant, predetermined, PMT voltage and amplifiergain settings. Preferably the excitation and emission wavelengths are423 and 525 nanometers, respectively, and no emission filter 139 is usedduring the measurement. Once this coefficient is measured for theseexcitation and emission wavelengths, it is used for measurements made atall excitation and emission wavelengths. Generally, emission andexcitation wavelengths are continuously selectable between 250 and 850nanometers and are settable in 1 nanometer increments.

PMT signal—The PMT signal is the measured intensity of the emission fromthe sample in question. It is determined from the PMT ADC counts bysubtracting the PMT ADC counts before the flash from the PMT ADC countsafter the flash (i.e., difference between post and pre counts for thesample).

Ref. sens. coeff.—The reference sensitivity coefficient is thesensitivity of the reference detector (i.e., detector 2409 in FIG. 24)as a function of excitation wavelength. Although this coefficient may bederived from an equation, in the preferred embodiment a look-up tablecontained within the system memory is used. Table 3 below provides thereference sensitivity coefficient for the preferred embodiment, as afunction of excitation wavelength. Coeffecients of intermediatewavelength values are obtained by linear interpolation.

TABLE 3 Excitation Reference Sensitivity Wavelength (nm) Coefficient 2000.25 300 0.25 400 0.50 500 0.77 600 0.95 700-850 1.07

Filter trans—The filter transmission value is the maximum transmissionvalue for a particular emission filter. Preferably these transmissionvalues are contained within a look-up table contained within the systemmemory. Table 4 below contains the maximum transmission value for eachof the emission filters shown in Table 2.

TABLE 4 Filter No. Filter Transmission 1 1.0  2 0.89 3 0.88 4 0.86 50.88 6 0.89 7 0.89 8 0.89 9 0.92 10  0.90 11  0.91 12  0.91 13  0.91 14 0.92 15  0.92

Ref. signal—The referance signal is the signal received from thereference detector which measureselative excitation light intensity(i.e., detector 2409 in FIG. 24).

e^(PMT voltage)—This value compensates for the gain of the PMT as afunction of voltage. In the preferred embodiment a predeterminedequation approximating the PMT manufacturer's published specificationsis used to establish this value.

PMT sens. coeff.—The PMT sensitivity coefficient is the sensitivity ofthe PMT as a function of emission wavelength. Although this coefficientmay be derived from an equation, in the preferred embodiment a look-uptable contained within the system memory is used. Table 5 below providesthe PMT sensitivity coefficient for the preferred embodiment, as afunction of emission wavelength. The values contained in Table 5 aretaken from the manufacturer's published spectral response of the PMTused in the system. Coefficients for intermediate wavelength values areobtained by linear interpolation.

TABLE 5 Emission Wavelength PMT Sensitivity (nm) Coefficient 300 0.6 400 0.7  500 0.95 600 0.78 700 0.54 800 0.4  900 0.27

In addition to the above, a calibration routine can be used to improvethe accuracy associated with changes in the PMT voltage. Normally thevoltage versus sensitivity characteristic of the PMT is an exponentialfunction. Unfortunately, 2 percent fit errors are not uncommon with avoltage change. In a preferred embodiment of the invention these errorsare eliminated by making small changes in the PMT voltage and measuringthe actual voltage versus gain curve for a given PMT under measuredconditions. These measurements may be made at the preselected emissionwavelength of interest and desired light intensity level by adjustingthe excitation wavelength suitably near the emission wavelength toprovide the desired intensity of reflected (and Raleigh scattered) straylight from low reference 1121.

Further refinement can be achieved through the use of a PMT matchingcoefficient. The matching coefficient is intended to compensate forvariations in PMT sensitivity as the voltage of the PMT is changed, forexample during incremental voltage changes when the system is operatingin the automated emission or excitation spectrum mode. The matchingcoefficient is defined as the calculated RFU at the first voltagedivided by the calculated RFU at the second voltage. In at least oneembodiment a table of matching coefficients is determined which can thenbe applied to subsequent sample scans.

Operational Modes

When one or more samples in a multi-assay plate are irradiated and theresulting fluorescent light collected and quantitated the plate is saidto be “read.” FIG. 18 illustrates the algorithm used when a plate isread using the automatic mode described briefly above. The first step,regardless of the mode of operation, is for the user to enter or selectthe various testing parameters (step 1801). For example, the user wouldselect the mode (e.g., automatic, manual) and depending upon the mode,other parameters such as the excitation wavelength, the emissionwavelength, the number of flashes per well, the emission cutoff filter,etc. The upper voltage for the PMT is then automatically determined bymeasuring the PMT signal of the low reference (step 1803). Based on theexperimentally determined voltage, medium and low PMT voltages are thenselected (step 1805). Although the preferred embodiment of the inventionutilizes three voltage levels, both fewer and greater numbers of voltagelevels can be used.

After the PMT voltages have been set, the PMT signal is measured for ahigh reference (step 1807) at the wavelengths specified for the highreference in order to determine the PMT calibration coefficient (step1809). High reference 1123 is a stable fluorescent material such as canbe obtained from Spec Check of Fullerton, Calif. and is contained incarriage 1101 as shown in FIG. 11.

The next step is to read the sample plate, assuming that the userselected a sample plate rather than a cuvette for analysis in step 1801.Prior to reading the plate, the system determines if the user hadinitially selected more than a single flash per sample well (step 1811).If the user had initially selected a single flash per well, the sampleplate and optics are moved to the first location, i.e., w=n (step 1813)and the sample well is read with a single flash from flash lamp 103(step 1815). During the first scan of the sample plate the PMT is set atthe high voltage, assuming that the user has not pre-selected a specificPMT voltage. In step 1815 four ADC gain conversions are calculated usinggain amplifiers 1115-1118 for the sample measurement. Gain multiplexer1111 selects the output from the gain amplifier that has the highestgain and yet is below the saturation level, passing the selected valueon to system processor 1101 for further processing. If none of theoutputs for a particular sample well are below the saturation level(step 1817), the sample well is tagged for further analysis bytemporarily storing the location of the well in memory.

After a sample well has been tested, the system determines whether theentire sample plate has been tested (step 1821). If there are moresample wells to be tested, the sample location is changed, i.e., n isincreased by 1 (step 1823) and the next sample well is tested. If all ofthe sample wells have been analyzed, the system determines whether ornot any sample wells were tagged (step 1825). If there are no taggedsample wells, the data is processed (step 1827). If there are any taggedsample wells, the system determines whether there is a lower PMT voltageavailable (step 1829) and if there is, the PMT voltage is decremented(step 1831) and the sample reading process starts over. Although all ofthe sample wells can be read at the lower PMT voltage, in the preferredembodiment only those samples that were previously tagged are read atthe lower voltage. If the system determines at step 1829 that there areno lower PMT voltages available, the data are processed (step 1833),noting those samples in which the PMT was saturated for all voltages.During the data processing steps (i.e., steps 1827 and 1833), preferablya table of RFU values for all of the sample wells is determined,although the data can be calculated and provided to the user in otherformats (e.g., plots, graphs, etc.). The intensity values for the wellsare normalized in accordance with the differences in PMT voltage.

If step 1811 determines that more than 1 flash has been selected, theprocedure for sample testing is basically the same as for the singleflash process with one notable exception. During sample testing, eachsample well is illuminated by flash lamp 103 with the preset number offlashes (step 1835). The data from these readings are then averaged(step 1837). In the preferred embodiment of the invention, during step1835 if the PMT saturates after the first flash, the preset number offlashes is over-ridden for the sample well in question, limiting thenumber of flashes to one.

FIG. 19 illustrates an alternative approach to the technique shown inFIG. 18. As in the previous approach, the user first enters varioustesting parameters (step 1801). In addition, the user selects a PMTvoltage (step 1901). The user may be provided with three pre-selectedvoltages to chose from, or the user may be given a range of voltagesfrom which to select. The rest of the process is the same as thatdescribed with reference to FIG. 18 except that the PMT voltage is notlowered after all of the samples have been tested. Rather, after theplate is read, the data are simply processed (step 1903).

FIG. 20 illustrates a variation of the method illustrated in FIG. 18.The steps in this variation are the same as in the FIG. 18 illustratedmethod up to and including step 1816. In the approach shown in FIG. 18,at step 1816 if the system determines that the PMT is saturated (step1817) then the sample well is tagged for further analysis in asubsequent scan of the sample plate at a lower voltage. In contrast, inthe approach shown in FIG. 20, if the system determines that the PMT issaturated (step 1817) and that a lower PMT voltage is available (step2001), the PMT voltage is decremented (step 2003) and the sample isre-tested. Re-testing of the sample at incrementally lower PMT voltagescontinues until either the PMT is not saturated (step 2005) or there areno lower PMT voltages available (step 2007). At that time the systemdetermines whether or not there are any more samples to be tested (step2009). If more samples remain (step 2011) then the process repeatsitself for the next sample (step 2013). Once all of the samples havebeen tested (step 2015), the data are processed (step 2017) aspreviously described. The same technique of testing a sample at multiplePMT voltages, if required, prior to moving to the next sample is alsoused for samples in which more than 1 flash was selected.

FIG. 21 illustrates another approach that may be utilized by the presentinvention. In this approach, termed a fly-by technique, a cursory reviewof all of the samples within a sample well is performed prior to thedetailed analysis of the samples. As in the prior approaches, the userinitially enters the data required by the system (step 1801), includingthe selection of the fly-by approach. The system then sets the PMTvoltage at a low level (step 2101) and the gain at a low level (step2103). After measuring the high reference (step 1807) and setting thePMT calibration coefficient (step 1809), the signal from the firstsample well is read (step 2105). Based on this signal, the appropriatePMT voltage and gain setting for this sample are determined (step 2107)and recorded (step 2109). This process continues, sample by sample,until all of the sample wells have been tested (step 2111). Sampletesting then starts over (step 2113). For each sample the PMT voltageand gain are set according to the pre-determined values (step 2115) andthe sample is tested (step 2117). Once all of the samples have beentested (step 2119), the data is processed (step 2121).

FIG. 22 illustrates a slight variation of the method shown in FIG. 21.As in the previous example, all of the samples are initially tested witha single flash with low PMT voltage and gain settings in order todetermine the appropriate PMT voltage and gain for a specific sample. Inthis method, however, after the initial testing of all of the samples(step 2111) the samples are sorted according to the determined PMTvoltage and gain settings (step 2201). Thus all samples requiring PMTvoltage “x” and gain “y” are placed in one bin, etc. Sample testing thenstarts (step 2203) with the PMT voltage and gain set at a first setting(step 2205). After all of the samples that were identified in steps 2107and 2201 as requiring these PMT voltage and gain settings are tested(step 2207), the system determines whether there are samples requiringother PMT voltage and gain settings (step 2209). If some of the samplesremain untested (step 2211), the settings are changed (step 2213) andall of the samples requiring the new settings are tested. Once all ofthe samples have been tested the data are processed (step 2215).Alternatively, the samples are sorted only according to PMT voltagerequired in step 2201 (i.e., not according to gain). Subsequent stepsshown in FIG. 22 remain the same except that multiplexer 1611automatically selects the appropriate gain as previously described.

FIG. 23 illustrates the spectrum mode of analysis. To implement thisform of analysis, the user selects the spectrum mode during the initialdata entry step (step 2301). Once the spectrum mode is selected, theuser must input information in addition to that normally required (step2303). For example, the user may be required to input whether to scanexcitation or emission wavelength, starting and stopping wavelengths,and the size of the wavelength increments to be used. Additionally, theuser may be required to choose between inputting a specific PMT voltage,selecting a pre-determined voltage level (e.g., low, medium, high), orallowing the system to automatically determine the optimal voltagesetting. In the auto mode, although the voltage may be varied betweenthree levels as described above with reference to FIG. 18, preferablythe voltage is varied in 25 volt increments. In some embodimentsincrements of a different size can be selected by the user.

Typically in the spectrum mode the system still determines an upper PMTvoltage (step 1803) and measures the PMT signal for a high reference(step 1807) in order to determine the PMT calibration coefficient (step1809). The wavelength is then set at a first wavelength (step 2305) andeach of the sample wells is read at this wavelength until all of thesamples have been read (step 2307). The wavelength is then incremented(step 2309) and the samples are re-tested. This process continues untilall of the samples have been tested for all of the wavelengths ofinterest (step 2311). The data are then processed (step 2313) anddisplayed as either excitation or emission spectra for each sampletested.

As noted above, during the spectrum scan the PMT voltage can either beheld at a predetermined voltage or level, or the system can operate inan automatic mode where the voltage is optimized. In another variant ofthe spectrum scanning mode illustrated in FIG. 23 the spectrum for asample is determined prior to moving to the next sample. Thus, insteadof scanning (i.e., reading the plate) all of the sample wells at asingle wavelength, incrementing the wavelength, and repeating the platereads until all of the wavelengths have been scanned, each sample can beindividually scanned at the predetermined wavelength values.

Well Optics

The preferred embodiment uses mirrored optics to guide the source lightinto the selected wells of the multi-assay plate and to guide theemitted light out of the selected wells. The use of mirrored opticsenhances the sensitivity of the instrument while reducing thebackground.

FIG. 24 is a schematic representation of the mirrored optics used toilluminate sample well 125 of a multi-assay plate and to receive theemitted light from the well. The source light passes through fiber 119to an elliptical focussing mirror 2401. The reflected light followsoptical paths 2403 to a sample well 125. A portion of the source lightis reflected by beamsplitter 2407 into a reference detector 2409.Detector 2409 is used to monitor the output of source 103, therebyallowing the intensity of the excitation beam to be normalized. In orderto illuminate sample well 125, thereby stimulating the emission offluorescence, phosphorescence, and/or scattered light, the lightreflected by mirror 2401, and transmitted through (i.e., not reflected)beamsplitter 2407, passes through an aperture 2411 in a secondelliptical focussing mirror 2413. Preferably less than 10 percent of thelight in optical paths 2403 is allowed to strike mirror 2413. The lightemitted by the sample within well 125 is reflected by mirror 2413 andfocussed into detection fiber 129. Specularly reflected light fromliquid meniscus 2421 of a liquid sample in well 125 and from the wellbottom surface 2419 (e.g., the light following optical path 2415) istrapped by one or more light traps 2417 which are constructed ofanodized aluminum. Preferably mirrors 2401 and 2413 are ellipticalfocusing mirrors, although they may be flat, spherical, or otherwisecurved. It will be appreciated by those of skill in the art that mirror2401 can be replaced by a lens, the lens either being separate from, orattached directly to, the end of fiber 119. The light from the lens, aswith mirror 2401 in the preferred embodiment, passes through aperture2411 of mirror 2413 prior to impinging upon the sample.

Significantly, aperture 2411 in mirror 2413 allows any preselectedwavelength of excitation light, which in the preferred embodiment isselected from the range of 250 to 850 nanometers, to pass through theaperture and impinge on the samples contained in sample wells 125.Furthermore, aperture 2411 is relatively small compared to the surfacearea of mirror 2413. In the preferred embodiment, the area of aperture2411 is less than 10% of the area of mirror 2413. More generally, thearea of aperture 2411 is less than 50% of the area of mirror 2413.

The preferred embodiment of the invention advantageously allows greaterthan 90% of the available excitation light energy to impinge upon thesamples within sample wells 125. This embodiment also allows greaterthan 90% of the light emitted from the samples within a solid anglesubtended by the perimeter of mirror 2413 to be focused directly onto aphotodetector, or, in the preferred embodiment, focused onto collectionoptical fiber 129 which transmits the emitted light to detector 135.Thus the design gives exceptionally efficient utilization and collectionof light energy while also allowing wide flexibility in the range ofoperable wavelengths.

FIG. 25 is an exploded view of optical scanning head 1201. FIGS. 26 and27 illustrate an upper and lower view, respectively, of the assembledhead 1201. Optical source fiber 119 (not shown in this view) ispreferably a one millimeter diameter optical fiber. Fiber 119 isattached to head block 2501 such that the source light passes through anaperture 2503 within block 2501. The image of source 103 projected byfiber 119 is magnified and focused a distance 2× in the sample well by areflective surface 2505 of mirror 2401. The focussed beam passes throughan aperture 2507 within block 2501. Beamsplitter 2407, preferably apiece of silica, is fitted within aperture 2507 such that the reflectedportion of the source light is detected by detector 2409 fitted to anoptical head cover plate 2509. The portion of the focussed beam notreflected by beamsplitter 2407 passes through aperture 2411 of detectionmirror 2413. Due to the source beam passing through mirror 2413, thismirror can be located very close to the sample well, thus enabling ahigh collection numerical aperture (i.e., NA).

Mirror 2413 is shown in greater detail in FIG. 28. This mirror has areversed magnification as compared to source mirror 2401. Thus fiber119, with a nominal 1 millimeter diameter, corresponds to a 2 millimeterexcitation beam at the focal point. This excitation beam generates a 360degree solid angle emission beam, a portion of which is collected bymirror 2413 and translated into a 4 millimeter image that is projectedonto the entrance aperture of emission fiber 129. A small fraction(i.e., less than 10%) of the emission light passes through aperture 2411and is not collected at emission fiber 129. The ratio of excitationlight power passing through aperture 2411 to that in the emission beamcollected by mirror 2413 for a high fluorescent sample is small,generally between 0.1 and 50 percent and preferably between 1 and 5percent (i.e., emission collection efficiency also generally variesbetween 0.1 and 50% and preferably between 1 and 5%).

Approximately a 4 millimeter diameter image is projected into collectionfiber 129 which is preferably 4 millimeters in diameter. Although thisis not the only focal geometry that can be used with the presentinvention, it is preferred since the 1 millimeter excitation apertureformed by optical fibers 119 corresponds to the maximum energy densityattainable with a xenon source/monochromator combination with anapproximately 10 nanometer bandpass. Similarly, the 4 millimeteremission aperture 141 corresponds to the typical available aperture fora PMT detector/monochromator combination with the desired 10 nanometerbandpass. The preferred focal geometry also provides for high emissioncollection efficiency.

In addition to the above noted benefits, apertures 2507 and 2411 help tocreate a well defined excitation beam, blocking a halo from forming nearthe beam. Without the aperture a portion of the halo is typicallyreflected, for example by the walls of the well, contributing tounwanted stray excitation light collected by the collection optics andpassed to detector 135. The uniform excitation beam diameter throughoutthe sample path insures uniformity of detection sensitivity within thesample. Furthermore, as the beam is substantially dispersed rather thantightly focussed to a small spot, inhomogeneous sample photodegradationis minimized.

In the embodiment of the invention illustrated in FIG. 24, the opticalpaths 2403 are not normal to bottom surface 2419 of sample wells 125(and are not normal to the support surfaces 1301 of carriage 1101 whichsupports the multi-assay plate having sample wells 125). Rather, theseoptical paths are canted by an angle θ with respect to the normal.Generally angle θ is between 0 and 30 degrees, and preferably angle θ isbetween 8 and 12 degrees. The cant of the optical paths allowsreflection of the excitation light beam off of bottom surface 2419 aswell as sample meniscus 2421, so as to miss detection mirror 2413.Instead these reflections impinge on light trap 2417. Light trap 2417 ispreferably a light absorption baffle. If surfaces 2419 of sample wells125 are clear, the excitation beam passes through the surface and iseither absorbed or reflected away, for example with a black polishedreflector (e.g., black glass) as illustrated by component 1119 of FIG.11. Alternatively, angle θ may be set at 0 degrees, i.e., normal tobottom surface 2419. A cant of 0 degrees is generally preferably forvery small sample wells 125 to minimize light scattering from welledges. This approach may also be preferable for absorption measurementsin which the source light passes completely through the sample and isdetected by a detection system coupled to the bottom surface ofmulti-assay plate 111 (see FIG. 1).

FIG. 29 illustrates an alternative embodiment of the invention. In thisembodiment the excitation beam is projected into sample well 125 throughits bottom surface. Similarly, the detected emission from the samplewell is also detected through the bottom surface. In order to minimizestray light background from the excitation source entering the detectionassembly, the excitation beam is at an angle to the normal to the bottomsurface of the well. A cuvette reading embodiment is illustrated in FIG.30. In this embodiment the excitation beam and the emission beam aresubstantially orthogonal, thereby minimizing the background.

Besides canting the optical paths by an angle θ as described above withreference to FIG. 24, preferably the system is further optimized bymoving the intersection of the optical path of excitation beam 2403 anda plane defined by the top of the sample well away from the samplewell's center line. Moving the optical path off center helps to reducethe amount of excitation light that reflects off of sample upper surface2421 and enters into detection fiber 129. Generally the offset from thecenter will be between 1 and 40 percent of the well diameter. The offsetis particularly effective when combined with canting the optical path byan angle θ as described above.

Several factors determine the distance that excitation beam 2403 shouldbe moved along axis 2423 to minimize undesired reflections. First, dueto the angular dependence of the reflections from surface 2421, theamount of cant angle θ is critical. Generally, greater values of cantangle θ will require greater offset distance from the center. Second,the curvature of meniscus 2421 is integral to determining where incidentbeam 2403 is reflected. The curvature depends upon the diameter andshape of the sample well and the characteristics (e.g., interfacialtension) of the sample with the walls of sample well 125.

FIG. 31 illustrates the dependence of the amount of excitation lightreflected into detector fiber 129 as a function of position. X-axis 3101shows the position of the incident excitation beam relative to a wellcenter 3103. Y-axis 3105 shows the PMT signal in arbitrary units. Curve3107 is an example of signal curve for a particular sample plate, samplematerial, etc. A peak 3109 of curve 3107 is due to the excitation beambeing reflected by the meniscus into the detection fiber. A minimum 3111of curve 3107 indicates the relative distance that the excitation beamshould be moved away from well center 3103 in order to minimizereflections and thus optimize the signal to background ratio.

In at least one embodiment of the invention, a look-up table isgenerated that provides the optimal position of the sample well relativeto the excitation beam for a variety of commonly used sample plates. Touse the look-up table the user enters the type of sample plate and thesystem automatically adjusts the well position. Alternately, a user canfirst set all of the various system parameters such as sample platetype, excitation beam cant angle, etc. and then run a calibration runusing a background reference sample in order to determine the optimalwell position. Then the sample plate containing the unknowns (i.e.,samples) can be run using the same well position.

Well-Scanning

As previously described, and as shown in FIGS. 11-15, the primaryapplication for the present invention is in scanning multi-assay plates.In this scanning mode, either a single sample reading can be made persample well, or a predetermined number of sample readings can be madeper sample well. If multiple readings per sample well are made,typically processor 1101 is programmed to center the excitation beam ata series of predetermined coordinates within the sample wells.Customarily the excitation and emission wavelengths are fixed.Wavelength scanning, however, may be performed in concert with thescanning mode to produce plots of spectral intensities (e.g., excitationand emission wavelengths) at each of the predetermined coordinates.

Frequently the fluorescent sample objects to be studied are biologicalcells (e.g., bacteria, algae, viruses, yeast, mammalian tissue cultures,etc.), subcellular organelles, or other biological constituents (e.g.,DNA, RNA, oligopeptides, proteins, etc.) that may be located at bottomsurface 2419 of sample wells 125. It is often advantageous to provide aliquid medium, such as water, isotonic saline, or a nutrient growthmedium, to maintain the viability of the sample objects (e.g., when thesample objects are living cells).

As previously noted, upper surface 2421 of the liquid medium within thesample wells typically forms a curved meniscus. The exact curvature ofthe meniscus depends upon the diameter and shape of sample well 125 andsuch characteristics as the interfacial tension between the samples andthe side walls of the wells. As a consequence of the curved meniscus, aportion of the excitation light may be reflected into the emissioncollection optics. The amount of such reflected excitation light dependsupon the angle of incidence of the excitation light beam with the curvedmeniscus. Thus as the coordinates of the excitation light beam arevaried within a sample well, the amount of excitation light collected bythe emission optics varies (see, for example, FIG. 31). Because of thevarying background intensity, it is difficult to accurately quantify thefluorescence measured at different X-Y coordinates within a singlesample well.

FIG. 32 is an illustration of the artifacts that can be generated duringwell-scanning. To generate the measurements shown in this figure, eightwells of a 24 well tissue culture microplate manufactured by Costar®(e.g., catalog number 3524) were scanned. The microplate is made ofpolystyrene which emits low levels of fluorescent light when excited at350 nanometers. Thus for purposes of this illustration, both long passexcitation filter 107 of filter wheel 108 and movable grating 109 wereselected to pass 350 nanometer light (i.e., filter number 2 of Table 1).Emission was measured at 440 nanometers with emission monochromatorgrating 137 set to focus 440 nanometer light onto detector 135. Emissioncutoff filter 139 in emission filter wheel 140 is optimally selected tobe a 435 nanometer longpass filter (i.e., filter number 3 of Table 2).

In this example, a total of 49 X-Y coordinates were examined in a 7 by 7square array for each sample well that was examined. To achieve thisscanning pattern, the excitation beam, with a diameter of about 3.5millimeters measured at the bottom surface of well 125, was positionedat 1.45 millimeter intervals. In FIG. 32, the patterns contained withinsquares 3201-3208 indicate the emission intensities for each samplinglocation, the relative positions of the individual sampling locationsshown as small squares. The intensity of the emission is indicated bythe relative position within a 7 level gray-scale, where an RFU equal to0 is indicated by a white square centered at the sampling location andan RFU greater than 200 is indicated by a black square centered at thesampling location. Intermediate levels of emission RFUs are indicated bycorresponding intermediate gray levels interpolated from white to blackas emission intensity values increase. Emission intensity values for thefour corner points in each 7 by 7 array are not shown because much ofthe excitation light for these X-Y coordinates intersects the plane ofbottom surface 2419 outside the side walls of sample wells 125.

As previously noted, only 8 of the 24 sample wells were tested. Thesample wells represented by emission intensity patterns 3201-3204contained 0.5 milliliters, 1.0 milliliters, 2.0 milliliters, and 2.5milliliters of water, respectively. The sample wells represented byemission intensity patterns 3205-3208 contained no liquid sample. Asshown, the emission intensity from dry wells 3205-3208 is relatively lowand uniform from each of the 45 scanned X-Y coordinates. In contrast,the emission intensity from sample wells 3201-3204 are much morenon-uniform at the equivalent scanned X-Y coordinates. Thus as shown,liquid in the sample wells creates a non-uniform pattern of apparentfluorescent emission, the level of pattern non-uniformity dependent uponthe water volume.

FIG. 33 is an illustration of the fluorescence emission intensity forthe same sample wells after the addition of an excitation light filter143 located between light source 103 and sample wells 125 (see FIG. 1).The exact location of additional excitation filter 143 is not criticalas long as retro-reflections of the filter are accounted for properly.As shown by FIG. 33, the additional excitation light filter to removestray light results in no significant change in the measuredfluorescence of dry wells 3205-3208, but greatly diminishes thenon-uniform emission pattern wells 3201-3204 containing liquid. Suchuniform emission patterns are highly desirable in well scanningapplications.

For the example illustrated in FIGS. 32-33, an additional filter 143 isadded to system 100 (see FIG. 1). Filter 143 is a short wavelength passfilter with a cutoff (i.e., the wavelength of 50% nominal transmission)of 400 nanometers. Such a filter can be obtained from the Andover Corp.of Salem, N.H. (e.g., model number 400FL07-25). Together with theselected 320 nanometer longpass filter 107 already employed (i.e.,filter number 2 of Table 1), the two filters comprise a wide-bandbandpass filter. This wide-band band pass filter is substantially opaquebelow 320 nanometers and cuts on, i.e., becoming substantiallytransparent, between 320 nanometers and 400 nanometers. This filtercombination then cuts off, i.e., becoming substantially opaque, atwavelengths longer than 400 nanometers. Thus the effective pass band ofthis filter is 80 nanometers.

According to the invention, generally the band pass of the wide bandpassfilter, i.e., filters 107 and 143 in the present example, is betweenabout 10 and about 100 nanometers, and preferably between about 40 andabout 80 nanometers. For full coverage of the complete spectral range ofexcitation wavelengths (i.e., from about 250 to about 750 nanometers inthe preferred embodiment) a series of such bandpass filters is used.Preferably the long wavelength cutoff value increases in 10-80 nanometerpredetermined steps. More preferably, the steps are between about 25 andabout 50 nanometers. Step values of between about 10 and 35 nanometersoffer the advantage of improved elimination of stray light at everypredetermined excitation wavelength. Step values between 35 and 80nanometers offer the advantage of requiring fewer sets of filters tocover the complete spectral range of excitation wavelengths. Thussmaller steps give improved performance but at the cost of requiringmore bandpass filters to cover the desired range of wavelengths. Itshould be understood that although in the present example a pair offilters, i.e., filters 107 and 143, are used in combination to providethe desired pass band, a single bandpass filter can also be used.Additionally it should be understood that the filters of the inventionmay be interference filters, colored glass filters, Wrattan filters, orother types of bandpass filters well known to those skilled in the art,used singly, or in combination to achieve the desired wavelength passband.

In the preferred embodiment of the invention, filters 107 within filterwheel 108 are bandpass filters. Additionally, filter wheel 108 isdesigned to include 8 filter positions. The preferred filters and theirrespective bandpasses to cover the excitation wavelength range of250-850 nanometers are given in Table 6.

TABLE 6 Filter No. Filter Characteristics 1  70 nm bandpass centered at280 nm 2  70 nm bandpass centered at 340 nm 3 360 longpass & 450shortpass 4 435 longpass & 500 shortpass 5 475 longpass & 550 shortpass6 530 longpass & 600 shortpass 7 570 longpass & 650 shortpass 8 630longpass

The examples illustrated above use bandpass filters in series with asingle excitation monochrometer to eliminate stray excitation light asshown in FIG. 1. A preferred alternative illustrated in FIG. 34 utilizesa double monochrometer on the excitation side of the scanningfluorometer to reduce stray light. In this embodiment, light from lightsource lamp 103 passes through one or more apertures 105 before beingdiffracted by grating 109 housed within a first monochrometer chamber3401. The light then passes through an exit slit 3403 prior to beingdirected into a second monochrometer chamber 3405 where the light isdiffracted by a second diffraction grating 3407. As in the previousembodiment, source light (in this example from source 103 andmonochrometer chambers 3401 and 3405) passes into a selected fiber ofoptical fibers 119, the selection of which is preferably determinedusing optical shutter 121. If desired, a filter 3409 within a filterwheel 3411 can be used in either monochrometer chamber 3401,monochrometer 3405, or both, to further enhance the reduction of strayexcitation light. The reduced level of stray excitation light isparticularly important for well scanning across open wells of amulti-assay plate having liquid samples where the excitation light isdirected into liquid samples through a gas/liquid (e.g., air/water)interface.

In yet another embodiment of the invention dual emission monochrometers3501 and 3503 are used, either alone as illustrated in FIG. 35, or incombination with dual excitation monochrometers such as thoseillustrated in FIG. 34. In the illustrated dual emission monochrometerembodiment, light emitted from wells 125 or cuvette 115 is directedalong a selected optical fiber of fibers 129, passing into firstemission monochrometer 3501 via optical shutter 133. Preferably opticalshutter 133 also acts as an entrance slit. The light strikes firstemission grating 137 and then exits the housing of the first emissionmonochrometer prior to passing through entrance slit 3505 into secondemission monochrometer 3503. Within monochrometer 3503 the light beam isdiffracted by a second emission grating 3507 before exiting slitaperture 141 and entering detector 135. As in the previous embodiment,if desired filters 3509 within one or more filter wheels 3511 can beused to further reduce the stray light.

Time Tagging

Time tagging refers to a method of monitoring the progression of areaction or of tracking a series of measurements. Therefore this processhas greatest utility when the material or composition underinvestigation is relatively unstable. For example, in a test todetermine the absorption properties of a series of materials of slightlydiffering composition, time tagging is typically not required, assumingthat the absorption properties of the materials do not change with time.In contrast, in a luminescence investigation where the luminescence of amaterial is initiated by the addition of one or more reactants, it maybe critical to record the time that the luminescence is measured withrespect to the time that the reactants were added. Furthermore, if theluminescent properties of a series of compositions are being compared, avalid comparison may require that the time sequence for each individualcomposition be recorded. As the number of samples under comparison isincreased, for example by using a multi-assay plate with a large numberof sample wells, monitoring the reaction kinetics as a function of timebecomes increasingly important. Particularly important is the exactamount of time passed between preparation and characterization of eachsample. Also, sequential repeating of the characterization processpermits kinetic analysis of the processes occurring in the one, or more,samples contained in the wells of a multi-assay plate.

In the preferred embodiment of the invention, the instrument can beoperated in several different time tagging modes. Although in thepreferred embodiment internal processor 1101 performs the time taggingfunction, a separate internal processor or an external processor mayalso perform this function.

The simplest mode of time tagging is to simply assign a single timepoint for an entire sample series. In this mode a first time is recordedthat is representative of the initiation of the experiment. This timepoint can represent, for example, the addition of a reactant to eachmaterial within the sample series. A second time is then recorded whenthe sample series is characterized (e.g., by measuring fluorescence,luminescence, or absorption). If the sample series is characterizedrepeatedly over time, each time the series is characterized a timeassociated with the characterization may be recorded. Thus each of thematerials within the series can be characterized as a function of time.Although this mode of time tagging is adequate for many investigations,it does not take into account variations encountered at the onset of theexperiment, for example due to the addition of the reactants in asequential rather than simultaneous fashion. Nor does this mode takeinto account any time lag associated with the measurement process. Thistime lag can be substantial for a multi-assay plate with a large numberof sample wells (e.g., 364 or 1500 sample wells).

In a second time tagging mode illustrated in FIGS. 36 and 37, although asingle time is used for an entire multi-assay plate, the discrepanciesbetween samples are substantially eliminated. As shown, both a samplepreparation system 3601 and a sample characterization system 3603 arecontrolled by a processor 3605. Although in the preferred embodiment asingle processor 3605 is used to control both systems 3601 and 3603,multiple processors can be used as long as certain timing functions areprogrammable as described more fully below. Associated with processor3605 is a clock 3607. Clock 3607 can be either an internal or anexternal clock. Although in the preferred embodiment clock 3607 providesan actual time (e.g., 2:32 PM), clock 3607 can also provide a runningtime. In the latter mode clock 3607 simply provides the amount of timethat has passed between time tags. Also coupled to processor 3605 is amemory 3609, either internal or external to the processor, that recordsthe time tags associated with each sample. Memory 3609 can be eithervolatile or non-volatile and utilize any of a variety of well knownmedia (e.g., electronic, dynamic random access memory, magnetic media,capacitive and charge-storage systems, optical storage systems, etc.).Also attached to processor 3605 are one or more data presentationsystems such as a printer 3611, a plotter 3613, and/or a monitor 3615.

Sample characterization system 3603 is a scanning fluorometer such asdescribed above, preferably capable of measuring luminescence andabsorption as well as fluorescence. Sample preparation system 3601 maybe a robotically controlled, material preparation system such as iscommonly known by those of skill in the art. In such a system adispensing mechanism (e.g., micropipette, syringe, etc.) is typicallyattached to a 2- or 3-axis robotic stage. The dispensing system placesthe desired concentrations of each of the components into the individualsample wells of the multi-assay plate. The preparation system may alsoinclude means of mixing the components within the sample wells and meansof varying the environment of the multi-assay plate (e.g., temperature,pressure, etc.). Sample preparation system 3601 can either be separatefrom, or combined with, characterization system 3605.

The methodology of this embodiment of the invention is illustrated inFIG. 37. Initially a sample plate is inserted into sample preparationsystem 3201 (step 3701) and processor 3605 is programmed with the type,size, and well configuration for the selected plate (step 3703). Thisprogramming provides the system with sufficient information to determinethe locations of each of the sample wells within the plate.Alternatively, the system can utilize sensors or other means to locatethe sample well positions. Processor 3605 is then programmed with thequantities of each of the individual components to be dispensed into theindividual sample wells to create the desired compositions (step 3705).

Typically the initiation of each reaction within each sample well can beattributed to a single, controllable event such as the introduction of areactant. If the reaction is initiated by the introduction of multiplereactants, often it is possible to simultaneously introduce thereactants, thus still resulting in a single reaction initiation time.Although the present example, illustrated in FIG. 37, assumes a singlecritical reaction initiation point, the present embodiment can also beused with more complicated, multi-critical event reactions. In thiscase, however, accurate time tagging is employed at each critical stage.

As illustrated in FIG. 37, the reaction within the first sample well ofthe multi-assay plate is initiated at a time equal to x (step 3707).Time x is recorded by processor 3605. The reaction within the secondsample well is initiated at a second time equal to x+Δ (step 3709),where Δ is a known time interval. Similarly, the reaction within thethird sample well is initiated at a third time equal to x+2Δ (step3711). By spacing the reaction initiations at known, regular timeintervals (step 3713), only the first time, x, must be recorded. Afterpreparation of the sample multi-assay plate has been completed, themulti-assay plate is removed from sample preparation system 3601 andinserted into sample characterization system 3603, assuming that twodifferent systems are employed (step 3715). The composition in the firstsample well is then characterized at a time equal to y (step 3717), withtimey being recorded by processor 3605. The second composition ischaracterized at a second time equal to y+Δ (step 3719), the thirdcomposition is characterized at a third time equal to y+2Δ (step 3721),and each subsequent composition is characterized using the same, known,regular time intervals (step 3723). Therefore assuming that the sameorder for sample preparation and sample characterization is used, thismethodology requires that only two times be recorded, a samplepreparation start time and a sample characterization start time,together with the time interval. This same methodology can be used withmultiple characterization runs by simply recording the start time ofeach characterization run and maintaining the same order and timeintervals.

In a specific example of this embodiment of the invention, a series oftwo primary component mixtures are first prepared in a multi-assay platewith 364 sample wells. A single reactant (i.e., a third component) isthen added to each of the sample wells. The first sample well isprepared at 8:00 AM, with subsequent samples being prepared at 30 secondtime intervals thereafter. Thus the 121^(st) sample is prepared at 9:00AM (i.e., 60 minutes for 120 samples, with a first sample at 8:00 AM).Using the present methodology, if the testing begins at 1:00 PM, the121^(st) sample will be characterized at 2:00 PM. As a consequence,although only 2 times were recorded, 10:00 AM and 1:00 PM, all of thesamples within the multi-assay plate can be directly compared since thetime intervals between the preparation of individual samples and thetime intervals between the characterization for individual samples isidentical. Furthermore, the preparation time and/or the characterizationtime for any particular sample can be easily calculated using the knownstart time and the known time interval. Obviously much shorter timeintervals may be employed for rapid analysis (e.g., 0.1 seconds orless).

In an alternative embodiment, sample preparation system 3601 and samplecharacterization system 3603 use two different processors. Thisembodiment offers the same benefits as the previous embodiments as longas the same time interval, Δ, is used by both processors and theinitiation times for the two processors can be correlated.

The system illustrated in FIG. 36 can also be used in an alternativeembodiment to apply a time tag to each individual sample. Preferably atime tag is applied at each critical sequence step for each samplewithin the multi-assay plate. For example, a time tag can be applied ateach preparation step as well as during each characterization step. Inaddition, time tagging can be applied as external variables such astemperature, humidity, gas pressure, gas type, etc. are altered.

FIG. 38 illustrates the methodology of this embodiment of the invention.When the reaction of sample 1 is initiated, a time t₁ is recorded (step3801). If necessary, multiple times can be recorded for the preparationof each sample, tagging each step or critical step of the process foreach sample. For example, although it may only be necessary to tag theintroduction of a reactant, it may be desirable to mark the introductionof each component of the composition, the starting and stopping times ofa composition mixing process, etc. After sample 1 is tagged, a time t₂is recorded for sample 2 (step 3803). This process continues until allof the samples to be prepared have been completed and tagged. The sameprocess is then used to record a time tag, t_(n), for eachcharacterization run made for each sample (step 3805).

This embodiment provides greater flexibility than the previouslydescribed embodiment since it is not necessary to maintain a constanttime interval between steps. Thus if some compositions are more complexthan others in the series and thus take longer to prepare, the timeinterval can be varied to accommodate the differences in preparationtime. Additionally, repetitive characterization runs can be made onselect samples of the series instead of having to adhere to the samesequence throughout the test. For example, if the user is testing 100different compositions, a preliminary characterization scan may showthat 90 percent of the samples are not worth further consideration. Theremaining 10 samples, however, can then be individually selected and amore thorough characterization performed on each of them. Once anexperimental run is complete, the data can either be stored for laterretrieval or immediately presented in a user-defined graphical ortabular format (e.g., absorption versus time; fluorescence versuscomposition versus time, etc.).

As will be understood by those familiar with the art, the presentinvention may be embodied in other specific forms without departing fromthe spirit or essential characteristics thereof. Accordingly, thedisclosures and descriptions herein are intended to be illustrative, butnot limiting, of the scope of the invention which is set forth in thefollowing claims.

What is claimed is:
 1. A sample characterizing instrument, comprising: asource of excitation light comprising: a lamp; a first scanningmonochromator; and a plurality of bandpass filters within a first filterwheel, wherein a bandpass associated with each of said plurality ofbandpass filters is between about 10 and about 100 nanometers; a sampletesting region; a detection assembly comprising: a detector; a secondscanning monochromator; and a plurality of optical filters within asecond filter wheel; an optical assembly coupled to said excitationlight source and coupled to said detection assembly, wherein saidoptical assembly directs said excitation light to said sample testingregion, and wherein said optical assembly directs sample light from saidsample testing region to said detection assembly; a carriage assemblycoupled to said optical assembly and to said sample testing region, saidcarriage assembly controlling the relative movement of said sampletesting region and said optical assembly to provide scanning of at leasta portion of said sample testing region; and a processor coupled to saidcarriage assembly, said excitation light source, and said detectionassembly, wherein said processor controls said scanning of said carriageassembly along a first scanning axis and a second scanning axis, whereinsaid processor controls an output wavelength of said excitation lightsource by controlling said lamp, said first scanning monochromator, andsaid plurality of bandpass filters, and wherein said processor controlsa detection wavelength of said detector by controlling said secondscanning monochromator and said plurality of optical filters.
 2. Thesample characterizing instrument of claim 1, wherein said bandpassassociated with each of said plurality of bandpass filters is betweenabout 40 and about 80 nanometers.
 3. The sample characterizinginstrument of claim 1, wherein a long wavelength cutoff valuecorresponding to each of said plurality of bandpass filters increases bypredetermined steps of about 10 to about 35 nanometers.
 4. The samplecharacterizing instrument of claim 1, wherein a long wavelength cutoffvalue corresponding to each of said plurality of bandpass filtersincreases by predetermined steps of about 35 to about 80 nanometers. 5.The sample characterizing instrument of claim 1, wherein said pluralityof bandpass filters are selected from the group consisting ofinterference filters, colored glass filters, or Wrattan filters.
 6. Thesample characterizing instrument of claim 1, said optical assemblyfurther comprising: a first reflector for directing said excitationlight toward said sample testing region; a second reflector forreflecting sample light from said sample testing region, said reflectedsample light detected by said detection assembly; and an aperture withinsaid second reflector, wherein said excitation light directed by saidfirst reflector passes through said aperture prior to impinging on saidsample testing region.
 7. The sample characterizing instrument of claim6, wherein a first optical axis corresponding to said second reflectoris offset by a first angle away from a normal to a bottom surface ofsaid sample testing region, and wherein a second optical axiscorresponding to said first reflector is offset by a second angle awayfrom said bottom surface normal.
 8. The sample characterizing instrumentof claim 6, wherein said first reflector and said second reflector areelliptical mirrors.
 9. The sample characterizing instrument of claim 1,further comprising a first optical fiber coupling said excitation lightsource to said optical assembly and a second optical fiber coupling saiddetection assembly to said optical assembly.
 10. The samplecharacterizing instrument of claim 1, further comprising: a heater; atemperature monitor; an air circulation system; and an enclosuresubstantially surrounding a sample plate holding fixture within saidsample testing region for use with a multi-assay plate, wherein ahumidity within said enclosure is in excess of 90 percent.
 11. A samplecharacterizing instrument, comprising: a source of excitation lightcomprising: a lamp; a first scanning monochromator; and a secondscanning monochromator; a sample testing region; a detection assemblycomprising: a detector; a third scanning monochromator; and a firstplurality of optical filters within a first filter wheel; an opticalassembly coupled to said excitation light source and coupled to saiddetection assembly, wherein said optical assembly directs saidexcitation light to said sample testing region, and wherein said opticalassembly directs sample light from said sample testing region to saiddetection assembly; a carriage assembly coupled to said optical assemblyand to said sample testing region, said carriage assembly controllingthe relative movement of said sample testing region and said opticalassembly to provide scanning of at least a portion of said sampletesting region; and a processor coupled to said carriage assembly, saidexcitation light source, and said detection assembly, wherein saidprocessor controls said scanning of said carriage assembly along a firstscanning axis and a second scanning axis, wherein said processorcontrols an output wavelength of said excitation light source bycontrolling said lamp, said first scanning monochromator, and saidsecond scanning monochromator, and wherein said processor controls adetection wavelength of said detector by controlling said third scanningmonochromator and said plurality of optical filters.
 12. The samplecharacterizing instrument of claim 11, said excitation light sourcefurther comprising a second plurality of optical filters within a secondfilter wheel.
 13. The sample characterizing instrument of claim 11, saidoptical assembly further comprising: a first reflector for directingsaid excitation light toward said sample testing region; a secondreflector for reflecting sample light from said sample testing region,said reflected sample light detected by said detection assembly; and anaperture within said second reflector, wherein said excitation lightdirected by said first reflector passes through said aperture prior toimpinging on said sample testing region.
 14. The sample characterizinginstrument of claim 13, wherein a first optical axis corresponding tosaid second reflector is offset by a first angle away from a normal to abottom surface of said sample testing region, and wherein a secondoptical axis corresponding to said first reflector is offset by a secondangle away from said bottom surface normal.
 15. The samplecharacterizing instrument of claim 13, wherein said first reflector andsaid second reflector are elliptical mirrors.
 16. The samplecharacterizing instrument of claim 11, further comprising a firstoptical fiber coupling said excitation light source to said opticalassembly and a second optical fiber coupling said detection assembly tosaid optical assembly.
 17. The sample characterizing instrument of claim11, further comprising: a heater; a temperature monitor; an aircirculation system; and an enclosure substantially surrounding a sampleplate holding fixture within said sample testing region for use with amulti-assay plate, wherein a humidity within said enclosure is in excessof 90 percent.
 18. A sample characterizing instrument, comprising: asource of excitation light comprising: a lamp; a first scanningmonochromator; and a first plurality of optical filters within a firstfilter wheel; a sample testing region; a detection assembly comprising:a detector; a second scanning monochromator; and a third scanningmonochromator; an optical assembly coupled to said excitation lightsource and coupled to said detection assembly, wherein said opticalassembly directs said excitation light to said sample testing region,and wherein said optical assembly directs sample light from said sampletesting region to said detection assembly; a carriage assembly coupledto said optical assembly and to said sample testing region, saidcarriage assembly controlling the relative movement of said sampletesting region and said optical assembly to provide scanning of at leasta portion of said sample testing region; and a processor coupled to saidcarriage assembly, said excitation light source, and said detectionassembly, wherein said processor controls said scanning of said carriageassembly along a first scanning axis and a second scanning axis, whereinsaid processor controls an output wavelength of said excitation lightsource by controlling said lamp, said first scanning monochromator, andsaid first plurality of optical filters, and wherein said processorcontrols a detection wavelength of said detector by controlling saidsecond scanning monochromator and said third scanning monochromator. 19.The sample characterizing instrument of claim 18, said detectionassembly further comprising a second plurality of optical filters withina second filter wheel.
 20. The sample characterizing instrument of claim18, said optical assembly further comprising: a first reflector fordirecting said excitation light toward said sample testing region; asecond reflector for reflecting sample light from said sample testingregion, said reflected sample light detected by said detection assembly;and an aperture within said second reflector, wherein said excitationlight directed by said first reflector passes through said apertureprior to impinging on said sample testing region.
 21. The samplecharacterizing instrument of claim 20, wherein a first optical axiscorresponding to said second reflector is offset by a first angle awayfrom a normal to a bottom surface of said sample testing region, andwherein a second optical axis corresponding to said first reflector isoffset by a second angle away from said bottom surface normal.
 22. Thesample characterizing instrument of claim 20, wherein said firstreflector and said second reflector are elliptical mirrors.
 23. Thesample characterizing instrument of claim 18, further comprising a firstoptical fiber coupling said excitation light source to said opticalassembly and a second optical fiber coupling said detection assembly tosaid optical assembly.
 24. The sample characterizing instrument of claim18, further comprising: a heater; a temperature monitor; an aircirculation system; and an enclosure substantially surrounding a sampleplate holding fixture within said sample testing region for use with amulti-assay plate, wherein a humidity within said enclosure is in excessof 90 percent.
 25. The sample characterizing instrument of claim 18,said excitation light source further comprising a fourth monochromator.